Evidence of cardiovascular protection by moderate alcohol and the polyphenol quercetin: Role of endothelial nitric oxide synthase

Heart disease remains the leading cause of death for men and women in the United States. Epidemiological studies suggest an inverse relationship between the intake of alcohol and/or polyphenols and the incidence of heart diseases. Despite the strong epidemiological data, molecular mechanisms for the benefits of alcohol and/or polyphenols remain elusive. Endothelial nitric oxide synthase (eNOS) is a key regulator of blood pressure through the generation of nitric oxide. Nitric oxide is an important vasodilator, an inhibitor of platelet aggregation, and a potent antiinflammatory molecule. Quercetin, the most abundant flavonoid in the human diet, and moderate alcohol have been implicated in a number of beneficial biological effects. We examined the effects of moderate alcohol and quercetin on eNOS. Alcohol pretreatment improved diastolic and vessel functions after ischemia-reperfusion injury and increased eNOS protein in rat aortic endothelium. Quercetin at 10 muM increased eNOS mRNA (approximately two fold) and protein (∼2.5-fold) in cultured human endothelial cells as evaluated by polymerase chain reaction and Western blots. Quercetin also increased eNOS mRNA in mice aorta 6 h after single treatment as evaluated by real time polymerase chain reaction and eNOS protein after 21 days of treatment as evaluated by both immunohistochemistry and Western blots. Experiments with actinomycin D, an inhibitor of transcription, suggested a role for transcription regulation. The full-length human eNOS promoter was amplified from genomic DNA and ligated to the promoterless, enhancerless luciferase plasmid; transient transfection of endothelial cells showed an increased promoter activity in the quercetin-treated cells compared to cells from controls. In an effort to advance our studies of the transcriptional regulation of eNOS by quercetin, the full-length human eNOS promoter was cloned into a lentiviral vector that expresses green fluorescent protein. Transient transfection of 293T cells with CL20c-eNOS-GFP was efficiently reproducible (75%); further treatment of eNOS-transduced cells showed an increase in green fluorescent protein expression in quercetin-, catechin-, and alcohol-treated cells compared to cells from controls. Transcriptional upregulation of eNOS by quercetin, in combination with the benefits of alcohol on cardiac functions, can explain, in part, the cardioprotective benefit of alcohol/polyphenol consumption.